SUMMARY (revised) The vitamin D hypothesis proposes that vitamin D regulates gastrointestinal homeostasis by multiple mechanisms and that changes in vitamin D status affect the composition of the microbiota, the development of the immune response, clearance of gastrointestinal infections and the ability to reinstate homeostasis following injury or infection. During the last grant cycle we determined several novel mechanisms by which vitamin D regulated T cells to control homeostasis in the gut. Our new preliminary data demonstrate that disruptions in the microbiota would inhibit vitamin D regulation of immune function and the metabolism of vitamin D by the host. These novel areas of investigation serve as the focus of the present application. The gastrointestinal immune system is a population of heterogeneous cells whose role is to maintain ignorance of the large number of antigens present in food as well as the microbiota. The microbiota in turn shapes the immune response. Germfree mice have reduced B cell IgG1, IgG2a and IgA responses, but hyper-IgE antibodies. Hyper-IgE is a symptom of dysbiosis and a failure of homeostasis. Vitamin D deficient and vitamin D receptor knockout mice have hyper-IgE and dysbiosis of the microbiota. In addition, following injury vitamin D regulates the T and B cell response in part through regulation of the microbiota. Perturbations of homeostasis, following gastrointestinal infection, of vitamin D deficient mice was severe and associated with the reduced production of antigen specific antibody responses. Not only was there an effect of vitamin D on the microbiota but the microbiota affected vitamin D metabolism. This proposal will focus on the effects of vitamin D on B cells and the microbial interactions that control the B cell response and microbial regulated tissue vitamin D metabolism. Our novel central hypothesis is that ?Vitamin D and the microbiota cooperate to regulate B cells and the vitamin D responsiveness of the host.? The three aims are: Aim 1: Determine the direct and indirect targets of vitamin D on B cells in vivo that are important for maintaining homeostasis, induction of antibody responses, and protection from GI infection. Aim 2: Determine which of the effects of vitamin D depend on the microbiota. Aim 3: Determine the role of the microbiota in the induction of 24,25(OH)2D by B cells.